The fungus Candida albicans is a commensal species of the human microbiota. It is also a frequently isolated opportunistic pathogen, responsible for a variety of mucosal and life-threatening systemic infections. The majority of these infections are a result of the ability of C. albicans cells to exist in biofilms ? resilient, surface-associated, communities of cells with unique properties. Many serious infections are highly correlated with implanted medical devices, which provide efficient substrates for biofilm formation. Recent studies have revealed that pheromone signaling is a novel mechanism promoting the formation of C. albicans biofilms. Pheromone signaling is well established to choreograph mating between C. albicans cells. Surprisingly, however, this program can also induce responding cells to become adherent and undergo biofilm development. Pheromones can drive biofilm formation in co-cultures of cells of opposite mating types. In addition, auto-activation of this pathway can even occur in single sex cultures, which similarly results in enhanced adherence of C. albicans cells and biofilm development. It is therefore clear that pheromones can play a pleiotropic role in driving biofilm formation by multiple C. albicans cell types, both by conventional and non-conventional pheromone signaling pathways. In this proposal, we seek to investigate the mechanism of pheromone-induced biofilm formation, and to address the potential for them to form in vivo using two mammalian models of biofilm infection. The two Aims of the proposal are: Aim 1. To define the mechanism of pheromone-induced biofilm formation. We will determine the mechanism by which pheromone signaling induces biofilm formation in C. albicans. Transcriptional profiling will be performed on a diverse set of clinical isolates during pheromone-induced biofilm formation, and target genes implicated in biofilm formation will be deleted by CRISPR and evaluated for their roles in this process. Aim 2. To determine the role of pheromone signaling on biofilm formation in vivo. We will determine if pheromone-signaling pathways lead to enhanced biofilm formation using two distinct mammalian models of biofilm formation, a rat catheter model and a denture stomatitis model. Together, completion of these experiments will reveal how pheromone-signaling pathways impact biofilm formation in C. albicans both in vitro and in vivo. Our approach is based on intriguing preliminary data that indicates that introduction of a mixture of two mating types results in enhanced biofilms in an animal model. If successful, this proposal has the potential to transform our understanding of C. albicans biofilm formation, and has direct clinical ramifications for treatment of fungal infections in patients. Overall, the combination of state- of-the-art genomic approaches, molecular genetics, high-resolution microscopy, microfluidic models, and two validated animal models designed to mimic human biofilm infections, provides a powerful combinatorial approach to studying this important clinical problem.